The threat presented by plant and agricultural diseases of natural origin lend urgency to the development of rapid, field-deployable diagnostic tools capable of detailed genetic analyses. While immunoassays have long been available as rapid field-ready assays in the form of dipstick-like hand-held devices Kohn, J., 1968, An immunochromatographic technique. Immunology 15(6): 863-5), the sequence variation and exponential amplification accessible to nucleic acid-based methods are widely recognized as enabling a greater level of specificity and sensitivity than typically associated with immunoassay (Andreotti, et al., 2003, Immunoassay of infectious agents. Biotechniques. 35(4): 850-859).
Additionally, DNA or RNA-based diagnostics offer the potential for more detailed insights to the presence of antibiotic resistance elements, virulence genes and other high-resolution genetic information, including pre-symptomatic host biomarkers of infection, that may not be assayed immunologically. Unfortunately, technical hurdles associated with the field deployment of the requisite nucleic acid manipulations, including sample preparation, amplification and detection, have confounded migration of nucleic acid-based assays from the laboratory to the field (Yang and Rothman, 2004, PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect Dis. 4(6): 337-48; Koch, W. H., 2004, Technology platforms for pharmacogenomic diagnostic assays. Nat Rev Drug Discov. 3(9): 749-761; Mackay, I. M., 2004, Real-time PCR in the microbiology laboratory. Clin Microbiol Infect. 10(3): 190-212; Cirino, et al., 2004, Multiplex diagnostic platforms for detection of biothreat agents. Expert Rev Mol Diagn. 4(6): p. 841-857).
Citrus is susceptible to a large number of diseases caused by plant pathogens. Economic losses due to plant diseases can be severe and are of particular concern in California and Florida, as well as in Brazil. In the state of Florida, citrus fruits, including oranges, grapefruit, tangelos, tangerines, limes, and other specialty fruits, are the state's largest agricultural commodity. Florida is the world's leading producing region for grapefruit and second only to Brazil in orange production. Florida produces over 80 percent of the United States' supply of citrus.
Citrus canker is a very serious disease affecting most commercial citrus varieties, and is caused by the bacterial pathogen Xanthomonas axonopodis pv. citri (“Xac”). The pathogen causes necrotic lesions on leaves, stems and fruit. Severe infections can cause defoliation, badly blemished fruit, premature fruit drop, twig dieback and general tree decline. Considerable regulatory effort is directed at preventing the spread of citrus canker because it is not present in all citrus-growing regions of the world where the climate is conducive to its development. Xac's presence, if detected, triggers immediate quarantines of areas with outbreaks, disrupting movement of fresh fruit. Eradication has typically involved burning uprooted trees.
There are several distinct types of citrus canker disease caused by various pathovars and variants of Xac. The Asiatic type of canker (Canker A), caused by a group of strains originally found in Asia, is by far the most widespread and severe form of the disease. This is the group of X. axonopodis pv. citri strains that causes the disease most referred to as Asiatic citrus canker. Minor genetic variation of citrus canker strains has been detected in the A strains in Florida and other citrus growing regions of the world, which may be exploited to identify their origin when introduced into new locations.
PCR-based methods for Xac detection have been described and are currently in use (Cubero, et al., 2001, Quantitative PCR method for diagnosis of citrus bacterial canker. Appl. Environ. Microbiol. 67:2849-2852; Hartung and Pruvost, 1993, Detection of Xanthomonas campestris pv. citri by the polymerase chain reaction. Appl. Environ. Microbiol. 59:1143-1148). The primers used for citrus canker diagnosis are based on the plasmid containing the pthA gene, the primary virulence element in all citrus canker strains (Hartung, et al., 1996, Phytopathology 86:95-101). Primers based on the pthA gene are available for detection of all canker strains in Florida and elsewhere (Cubero and Graham, 2002, Appl. Environ. Microbiol. 68:1257-1264). Unfortunately, these same primers generate a PCR product of the same size in Xanthomonads not currently thought to cause citrus canker, limiting their utility for real-world applications where contaminating microbial flora may generate a false positive using this assay (Cubero and Graham, 2002, Appl. Environ. Microbiol. 68:1257-1264). Additionally, the use of a plasmid derived sequences may be undesirable due to the increased potential for horizontal gene transfer.
Primers for the identification of Xac based upon the internally transcribed spacer between 16S and 23S rRNA genes have also been reported. Again, however, this set of primers generates an amplicon from X. axonopodis pv citri and not other Xanthomonads cultured from citrus tissue, but lacks specificity within the broader diversity found within Xanthomonas spp. resulting in false positive signals when challenged with some other Xanthomonads. These characteristics render such assays of utility only for the identification of Xac from among bacteria isolated and cultured from citrus tissue not for the detection and identification of bacteria on plant samples.
Additionally, all of the available assays rely on PCR technology and fluorescent detection of amplified nucleic acids, which requires the use of complex laboratory instrumentation and involve high per assay cost (Yang and Rothman, 2004, PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect Dis. 4: 337-348: Koch, W. H., 2004, Technology platforms for pharmacogenomic diagnostic assays. Nat Rev Drug Discov. 3: 749-761; Mackay, I. M., 2004, Real-time PCR in the microbiology laboratory. Clin Microbiol Infect. 10: 190-212; Cirino, et al., 2004, Multiplex diagnostic platforms for detection of biothreat agents. Expert Rev Mol Diagn. 4: 841-857). Existing technologies, therefore, are not amenable to field deployment, which remains a significant unmet need.
Citrus variegated chlorosis (CVC) is another major disease affecting citrus in Brazil and Florida, as well as other citrus growing regions (Chang et al., 1993, Curr. Microbiol. 27: 137-142). In Brazil, it was estimated to have been present in over one-third of the 200 million citrus trees in the state of Sao Paulo in 2001 (Brlansky et al., 2002, Plant Disease 86(11): 1237-39). CVC causes severe leaf chlorosis between veins. Infected citrus trees typically exhibit attenuated vigor and growth, and show abnormal flowering and fruit sets. Fruits in CVC-infected trees are often small, hard and of high acid content, thus rendering them unsuitable for markets or juice processing. As trees mature, the disease typically spreads from one limb to another, and eventually to the entire tree. Eradication measures are extreme, and include the removal of entire orchards upon a threshold level of infection (in Brazil, for example, that threshold is 30%).
CVC is caused by the bacterial pathogen Xylella fastidiosa (Xf), which also causes a number of other diseases in commercial crops, including Pierce's Disease in grapevines (Davis et al., 1978, Science 199: 75-77), alfalfa dwarf disease (Goheen et al., 1973, Phytopathology 63: 341-345), and leaf scorch disease or dwarf syndromes in numerous other agriculturally significant plants, including almonds, coffee, and peach (Hopkins, 1989, Annu. Rev. Phytopathol. 27: 271-290; Wells et al., 1983, Phytopathology 73: 859-862; De Lima, et al., 1996, Fitopatologia Brasileira 21(3)). Although many agriculturally important plants are susceptible to diseases caused by Xf, in the majority of plants Xf behaves as a harmless endophyte (Purcell and Saunders, 1999, Plant Dis. 83: 825-830). Strains of Xf are genetically diverse and pathogenically specialized (Hendson, et al., 2001, Appl. Environ. Microbiol 67: 895-903). For example, certain strains cause disease in specific plants, while not in others. Additionally, some strains will colonize a host plant without causing the disease that a different Xf strain causes in the same plant.
Xf is acquired and transmitted to plants by leafhoppers of the Cicadellidae family and spittlebugs of the Cercropidae family (Purcell and Hopkins, 1996, Annu. Rev. Phytopathol. 34: 131-151). Once acquired by these insect vectors, Xf colonies form a biofilm of poorly attached Xf cells inside the insect foregut (Briansky et al., 1983, Phytopathology 73: 530-535; Purcell et al., 1979, Science 206: 839-841). Thereafter, the insect vector remains a host for Xf propagation and a source of transmission to plants (Hill and Purcell, 1997, Phytopathology 87: 1197-1201). In susceptible plants, Xf multiplies and spreads from the inoculation site into the xylem network, where it forms colonies that eventually occlude xylem vessels, blocking water transport. Prior studies have suggested that the presence of as few as 100 Xf cells in an insect vector is sufficient to enable transmission of the agent to a susceptible plant (Hill and Purcell, 1995, Pytopathology 85: 209-212). The low titer of Xf that can confer infection presents a challenge for commonly deployed serological diagnostics, e.g. ELISA, which typically require titers in excess of 1000 cells/ml for reliable detection.
For the sensitivity required to detect Xf in both plant and insect vector tissues a molecular assay must be used. Available molecular assays for the detection of Xf rely upon PCR assay platforms, and are therefore limited in their utility and field-deployability (Rodrigues, et al., 2003, “Detection and diversity assessment of Xylella fastidiosa in field-collected plant and insect samples by using 16S rRNA and gyrB sequences.” Appl Environ Microbiol 69(7): 4249-55; Ciapina, et al., 2004, “A nested-PCR assay for detection, of Xylella fastidiosa in citrus plants and sharpshooter leafhoppers.” J Appl Microbiol 96(3): 546-51; Pooler, et al., 1997, “Detection of Xylella fastidiosa in potential insect vectors by immunomagnetic separation and nested polymerase chain reaction.” Lett Appl Microbiol 25(2): 123-6; Pooler, M. R. and J. S. Hartung, 1995, “Specific PCR detection and identification of Xylella fastidiosa strains causing citrus variegated chlorosis.” Curr Microbiol 31(6): 377-81). The development of a sensitive isothermal assay for Xf would increase the simplicity of Xf detection and provide a protocol easily adaptable to a field deployable detection system.